Live cell constructs for production of cultured milk product and methods using the same

ABSTRACT

This invention relates to live cell constructs for in vitro and/or ex vivo production of cultured milk products from mammary cells, methods of producing isolated cultured milk products from mammary cells, bioreactors for producing isolated cultured milk products, and cultured milk products.

CROSS-REFERENCE

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/958,407, filed on Jan. 8, 2020, and U.S. ProvisionalApplication No. 63/199,164, filed on Dec. 10, 2020, the contents of eachare incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 14, 2021, isnamed BMQ-001_SL.txt and is 29,487 bytes in size.

FIELD OF THE INVENTION

This invention relates to live cell constructs and methods using thesame for in vitro and/or ex vivo production of cultured milk productfrom cultured mammary cells.

BACKGROUND OF THE INVENTION

Milk is a staple of the human diet, both during infancy and throughoutlife. The American Academy of Pediatrics and World Health Organizationrecommend that infants be exclusively breastfed for the first 6 monthsof life, and consumption of dairy beyond infancy is a mainstay of humannutrition, representing a 700 billion dollar industry worldwide.However, lactation is a physiologically demanding and metabolicallyintensive process that can present biological and practical challengesfor breastfeeding mothers, and milk production is associated withenvironmental, social, and animal welfare impacts in agriculturalcontexts.

The possibility of using mammalian cell culture to produce food hasgained increasing interest in recent years, with the development ofseveral successful prototypes of meat and sea food products fromcultured muscle and fat cells (Stephens et al. 2018 Trends Food SciTechnol. 78:155-166). Additionally, efforts are underway tocommercialize the production of egg and milk proteins using microbialexpression systems. However, this fermentation-based process relies onthe genetically engineered expression and purification of individualcomponents and is unable to reproduce the full molecular profile of milkor dairy.

The present invention overcomes shortcomings in the art by providinglive cell constructs and methods using the same for in vitro and/or exvivo production of cultured milk product from cultured mammary cells.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are live cell constructs,comprising: (a) a three-dimensional scaffold having an exterior surface,an interior surface defining an interior cavity/basal chamber, and aplurality of pores extending from the interior surface to the exteriorsurface; (b) a matrix material disposed on the exterior surface of thethree-dimensional scaffold; (c) a culture media disposed within theinterior cavity/basal chamber and in fluidic contact with the internalsurface; and (d) an at least 70% confluent monolayer of polarizedmammary cells disposed on the matrix material, wherein the mammary cellsare selected from the group consisting of: live primary mammaryepithelial cells, live mammary myoepithelial cells, live mammaryprogenitor cells, live immortalized mammary epithelial cells, liveimmortalized mammary myoepithelial cells, and live immortalized mammaryprogenitor cells. In some embodiments, the polarized mammary cellscomprise an apical surface and a basal surface. In some embodiments, thebasal surface of the mammary cells is in fluidic contact with theculture media. In some embodiments, at least 70%, at least 80%, at least90%, at least 95%, at least 99%, or 100% of the mammary cells arepolarized in the same orientation. In some embodiments, the monolayer ofpolarized mammary cells is at least 70% confluent, at least 80%confluent, at least 90% confluent, at least 95% confluent, at least 99%confluent, or 100% confluent. In some embodiments, the mammary cellscomprise a constitutively active prolactin receptor protein. In someembodiments, the culture medium comprises a carbon source, a chemicalbuffering system, one or more essential amino acids, one or morevitamins and/or cofactors, and one or more inorganic salts. In someembodiments, the culture medium further comprises prolactin. In someembodiments, the matrix material comprises one or more extracellularmatrix proteins. In some embodiments, the three-dimensional scaffoldcomprises a natural polymer, a biocompatible synthetic polymer, asynthetic peptide, a composite derived from any of the preceding, or anycombination thereof. In some embodiments, the natural polymer iscollagen, chitosan, cellulose, agarose, alginate, gelatin, elastin,heparan sulfate, chondroitin sulfate, keratan sulfate, and/or hyaluronicacid. In some embodiments, the biocompatible synthetic polymer ispolysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate,polyvinyl alcohol, sodium polyacrylate, an acrylate polymer, and/orpolyethylene glycol.

Disclosed herein, in certain embodiments, are methods of producing anisolated cultured milk product from mammary cells, the methodcomprising: (a) culturing a live cell construct in a bioreactor underconditions which produce the cultured milk product, said live cellconstruct comprising: (i) a three-dimensional scaffold having anexterior surface, an interior surface defining an interior cavity/basalchamber, and a plurality of pores extending from the interior surface tothe exterior surface; (ii) a matrix material disposed on the exteriorsurface of the three-dimensional scaffold; (iii) a culture mediadisposed within the interior cavity/basal chamber and in fluidic contactwith the internal surface; and (iv) an at least 70% confluent monolayerof polarized mammary cells disposed on the matrix material, wherein themammary cells are selected from the group consisting of: live primarymammary epithelial cells, live mammary myoepithelial cells, live mammaryprogenitor cells, live immortalized mammary epithelial cells, liveimmortalized mammary myoepithelial cells, and live immortalized mammaryprogenitor cells; and (b) isolating the cultured milk product. In someembodiments, the polarized mammary cells comprise an apical surface anda basal surface. In some embodiments, the basal surface of the mammarycells is in fluidic contact with the culture media. In some embodiments,the bioreactor is an enclosed bioreactor. In some embodiments, thebioreactor comprises an apical compartment that is substantiallyisolated from the internal cavity/basal chamber of the live cellconstruct. In some embodiments, the apical compartment is in fluidiccontact with the apical surface of the mammary cells. In someembodiments, the cultured milk product is secreted from the apicalsurface of the mammary cells into the apical compartment. In someembodiments, the culture media substantially does not contact thecultured milk product. In some embodiments, the total cell density ofmammary cells within the bioreactor is at least 10¹¹. In someembodiments, the total surface area of mammary cells within thebioreactor is at least 1.5 m². In some embodiments, the culture mediumcomprises a carbon source, a chemical buffering system, one or moreessential amino acids, one or more vitamins and/or cofactors, and one ormore inorganic salts. In some embodiments, the matrix material comprisesone or more extracellular matrix proteins. In some embodiments, thescaffold comprises a natural polymer, a biocompatible synthetic polymer,a synthetic peptide, a composite derived from any of the preceding, orany combination thereof. In some embodiments, the natural polymer iscollagen, chitosan, cellulose, agarose, alginate, gelatin, elastin,heparan sulfate, chondroitin sulfate, keratan sulfate, and/or hyaluronicacid. In some embodiments, the biocompatible synthetic polymer ispolysulfone, polyvinylidene fluoride, polyethylene co-vinyl acetate,polyvinyl alcohol, sodium polyacrylate, an acrylate polymer, and/orpolyethylene glycol. In some embodiments, the culturing is carried outat a temperature of about 27° C. to about 39° C. In some embodiments,the culturing is carried out at a temperature of about 30° C. to about37° C. In some embodiments, the culturing is carried out at anatmospheric concentration of CO₂ of about 4% to about 6%. In someembodiments, the culturing is carried out at an atmosphericconcentration of CO₂ of about 5%.

Disclosed herein, in certain embodiments, are bioreactors, comprising:(a) an apical compartment comprising a cultured milk product; and (b) atleast one live cell construct comprising: (i) a three-dimensionalscaffold having an exterior surface, an interior surface defining aninterior cavity/basal chamber, and a plurality of pores extending fromthe interior surface to the exterior surface; (ii) a matrix materialdisposed on the exterior surface of the three-dimensional scaffold;(iii) a culture media disposed within the interior cavity/basal chamberand in fluidic contact with the internal surface; and (iv) an at least70% confluent monolayer of polarized mammary cells disposed on thematrix material, wherein the mammary cells are selected from the groupconsisting of: live primary mammary epithelial cells, live mammarymyoepithelial cells, live mammary progenitor cells, live immortalizedmammary epithelial cells, live immortalized mammary myoepithelial cells,and live immortalized mammary progenitor cells; wherein the apicalsurface of the mammary cells is in fluidic contact with the apicalcompartment. In some embodiments, the total cell density of mammarycells within the bioreactor is at least 10¹¹. In some embodiments, thetotal surface area of mammary cells within the bioreactor is at least1.5m².

Disclosed herein, in certain embodiments, are live cell constructscomprising mammary cells that compartmentalize feeding of the cells andsecretion of cultured milk product.

Disclosed herein, in certain embodiments, are live cell constructscomprising, a scaffold having a top surface and a bottom surface; and acontinuous monolayer of (a) live primary mammary epithelial cells, (b) amixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, and/or (c) liveimmortalized mammary epithelial cells on the top surface of thescaffold, the continuous monolayer of (a) live primary mammaryepithelial cells, (b) mixed population of live primary mammaryepithelial cells, mammary myoepithelial cells and mammary progenitorcells, and/or (c) immortalized mammary epithelial cells having an apicalsurface and a basal surface (e.g., the cells form a polarized andconfluent cell monolayer), wherein the construct comprises an apicalcompartment above and adjacent to the apical surface of the continuousmonolayer of the (a) live primary mammary epithelial cells, the (b)mixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, and/or the (c)immortalized mammary epithelial cells and a basal compartment below andadjacent to the bottom surface of the scaffold.

Disclosed herein, in certain embodiments, are methods of producing milkin culture, the method comprising culturing the live cell construct ofthe present invention, thereby producing milk in culture.

Disclosed herein, in certain embodiments, are methods of making a livecell construct for producing milk in culture, the method comprising (a)isolating primary mammary epithelial cells, myoepithelial cells and/ormammary progenitor cells from mammary explants from mammary tissue(e.g., breast, udder, teat tissue), biopsy sample, or raw breastmilk, toproduce isolated mammary epithelial cells, myoepithelial cells andmammary progenitor cells; (b) culturing the isolated primary mammaryepithelial cells, myoepithelial cells and mammary progenitor cells toproduce a mixed population of primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells; (c) cultivating themixed population of (b) on a scaffold, the scaffold having an uppersurface and lower surface, to produce a polarized, continuous (i.e.,confluent) monolayer of primary mammary epithelial cells, myoepithelialcells and mammary progenitor cells of the mixed population on the uppersurface of the scaffold, wherein the polarized, continuous monolayercomprises an apical surface and a basal surface, thereby producing alive cell construct for producing milk in culture.

Disclosed herein, in certain embodiments, are methods of making a livecell construct for producing milk in culture, the method comprising: a)isolating primary mammary epithelial cells, myoepithelial cells, and/ormammary progenitor cells from mammary explants from mammary tissue(e.g., breast, udder, teat tissue), biopsy sample, or raw breastmilk, toproduce isolated mammary epithelial cells, myoepithelial cells, and/ormammary progenitor cells; (b) culturing the isolated primary mammaryepithelial cells, myoepithelial cells, and/or mammary progenitor cellsto produce a mixed population of primary mammary epithelial cells,mammary myoepithelial cells and mammary progenitor cells; (c) sortingthe mixed population of primary mammary epithelial cells, myoepithelialcells, and/or mammary progenitor cells to produce a population ofprimary mammary epithelial cells; and (d) cultivating the population ofprimary mammary epithelial cells on a scaffold, the scaffold having anupper surface and lower surface, to produce a polarized, continuous(i.e., confluent) monolayer of primary mammary epithelial cells on theupper surface of the scaffold, wherein the polarized, continuousmonolayer comprises an apical surface and a basal surface, therebyproducing a live cell construct for producing milk in culture.

Disclosed herein, in certain embodiments, are methods of making a livecell construct for producing milk in culture, the method comprising (a)culturing immortalized mammary epithelial cells to produce increasednumbers of immortalized mammary epithelial cells; (b) cultivating theimmortalized mammary epithelial cells of (a) on a scaffold, the scaffoldhaving an upper surface and lower surface, to produce a polarized,continuous (i.e., confluent) monolayer of immortalized mammaryepithelial cells on the upper surface of the scaffold, wherein thepolarized, continuous monolayer comprises an apical surface and a basalsurface, thereby producing a live cell construct for producing milk inculture.

Disclosed herein, in certain embodiments, are methods of producing milkin culture comprising, culturing a live cell construct comprising (a) ascaffold comprising an upper surface and a lower surface and acontinuous (i.e., confluent) polarized monolayer of live primary mammaryepithelial cells, a continuous polarized monolayer of a mixed populationof live primary mammary epithelial cells, mammary myoepithelial cellsand mammary progenitor cells, and/or a continuous polarized monolayer oflive immortalized mammary epithelial cells having an apical surface anda basal surface, wherein the continuous polarized monolayer of liveprimary mammary epithelial cells, the continuous polarized monolayer ofthe mixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells and/or the continuouspolarized monolayer of live immortalized mammary epithelial cells arelocated on the upper surface of the scaffold, (b) a basal compartmentand an apical compartment, wherein the lower surface of the scaffold isadjacent to the basal compartment and the apical surface of themonolayer of live primary mammary epithelial cells, the monolayer of themixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, and/or the monolayerof live immortalized mammary epithelial cells is adjacent to the apicalcompartment, wherein the monolayer of live primary epithelial mammarycells, the live primary epithelial mammary cells of the monolayer of themixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, or the monolayer ofimmortalized mammary epithelial cells secretes milk through its apicalsurface into the apical compartment, thereby producing milk in culture.

Disclosed herein, in certain embodiments, are methods of producing amodified primary mammary epithelial cell or an immortalized mammaryepithelial cell, wherein the method comprises introducing into the cell:(a) a polynucleotide encoding a prolactin receptor comprising a modifiedintracellular signaling domain, optionally wherein the prolactinreceptor comprises a truncation wherein position 154 of exon 10 has beenspliced to the 3′ sequence of exon 11; (b) a polynucleotide encoding achimeric prolactin receptor that binds to a ligand, which is capable ofactivating milk synthesis in the absence of prolactin; (c) apolynucleotide encoding a constitutively or conditionally activeprolactin receptor protein, optionally wherein the polynucleotideencodes a constitutively active human prolactin receptor proteincomprising a deletion of amino acids 9 through 187; (d) a polynucleotideencoding a modified (recombinant) effector of a prolactin proteincomprising (i) a JAK2 tyrosine kinase domain fused to a STATS tyrosinekinase domain; and/or (ii) a prolactin receptor intracellular domainfused to a JAK2 tyrosine kinase domain; (e) a loss of function mutationinto a circadian related gene PER2 (period circadian protein homolog 2);and/or (f) a polynucleotide encoding one or more glucose transportergenes GLUT1 and/or GLUT12, thereby increasing the rate of nutrientuptake at the basal surface of a monolayer of cells of the modifiedprimary mammary epithelial cell or immortalized mammary epithelial cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of the collection of milk for nutritional usefrom mammary epithelial cells grown as a confluent monolayer in acompartmentalizing culture apparatus in which either fresh or recycledmedia is provided to the basal compartment and milk is collected fromthe apical compartment. TEER, transepithelial electrical resistance.

FIG. 2 shows an example of polarized absorption of nutrients andsecretion of milk across a confluent monolayer of mammary epithelialcells anchored to a scaffold at the basal surface.

FIG. 3 shows an example micropatterned scaffold that provides increasedsurface area for the compartmentalized absorption of nutrients andsecretion of milk by a confluent monolayer of mammary epithelial cells.

FIG. 4 shows three examples of a hollow fiber bioreactor depicted as abundle of capillary tubes (top), which can support mammary epithelialcells lining either the external (top and lower left) or internal (lowerright) surface of the capillaries, providing directional andcompartmentalized absorption of nutrients and secretion of milk.

FIG. 5 exemplifies a cross-section of three-dimensional live cellconstruct. The construct is made up of a scaffold having an interiorsurface defining an interior cavity/basal chamber and an exteriorsurface. The interior cavity/basal chamber comprises cell culture media.A matrix material sits on top of the exterior surface of the scaffold.Pores transverse the scaffold from the interior surface to the exteriorsurface, allowing cell media to contact the basal surface of the cellsof the cell monolayer disposed on the matrix material.

FIG. 6 exemplifies a bioreactor for producing a cultured milk product.The bioreactor is made up of a live cell construct and an apicalchamber. The cell construct is made up of a scaffold having an interiorsurface defining an interior cavity/basal chamber and an exteriorsurface. The cavity comprises cell culture media. A matrix material sitson top of the exterior surface of the scaffold. Pores transverse thescaffold from the interior surface to the exterior surface, allowingcell media to contact the basal surface of the cells of the cellmonolayer disposed on the matrix material. The apical surface of thecells of the cell monolayer secrete the milk/cultured milk product intothe apical chamber. The apical chamber and the interior cavity/basalchamber are separated by the cell monolayer.

FIG. 7 exemplifies a live cell construct. The construct is made up of ascaffold having an interior surface defining an interior cavity/basalchamber and an exterior surface. The interior cavity/basal chambercomprises cell culture media. A matrix material sits on top of theexterior surface of the scaffold. Pores transverse the scaffold from theinterior surface to the exterior surface, allowing cell media to contactthe basal surface of the cells of the cell monolayer disposed on thematrix material.

DETAILED DESCRIPTION OF THE INVENTION

Milk is a nutrient-rich liquid food produced in the mammary glands ofmammals. It is a primary source of nutrition for infant mammals(including humans who are breastfed) before they are able to digestother types of food. Human milk is not merely nutrition. Rather, humanmilk contains a variety of factors with bioactive qualities that have aprofound role in infant survival and health. Natural milk contains manyother macronutrients, including proteins, lipids, polysaccharides andlactose. Milk consumption occurs in two distinct overall types: anatural source of nutrition for all infant mammals and a food product.

In almost all mammals, milk is fed to infants through breastfeeding,either directly or by expressing the milk to be stored and consumedlater. Early milk from mammals contains antibodies that provideprotection to the newborn baby as well as nutrients and growth factors.Breast milk is not a uniform, unvarying, constant, factory-made product;rather, it is a biological product produced by women with markedlyvarying genotypes, phenotypes, and diets. To add to the complexity, thecomposition of breast milk is influenced by a myriad of maternal,infant, and environmental factors. Human milk contains a rich array ofproteins, carbohydrates, lipids, fatty acids, minerals, and vitamins,but most of its disease-fighting potential comes from a plethora ofantibodies, leukocytes, hormones, antimicrobial peptides, cytokines,chemokines, and other bioactive factors.

Mammary epithelial cells (MECs) in culture have been previouslydemonstrated to display organization and behavior similar to thatobserved in vivo (Arevalo et al. 2016 Am J Physiol Cell Physiol. 310(5):C348-3 56; Chen et al. 2019 Curr Protoc Cell Biol. 82(1):e65). InArevalo et al., specific biomarkers of MEC populations were detected inimmortalized bovine mammary epithelial cells (BME-UV1) and immortalizedbovine mammary alveolar cells (MAC-T) cultured on adherent 2-D plates,ultralow attachment surface 3D microplates, and 3D plates coated withMatrigel. Additionally, in Chen et al., protocols are detailed forisolation and culture of human primary mammary epithelialstem/progenitor cells from human breast tissue and subsequent generationof mammospheres using 3D organoid culture on gelatin sponges andMatrigel matrices. However, neither Arevalo nor Chen attempted tostimulate the production of milk from these MEC cultures.

In particular, when grown on an appropriate extracellular matrix andstimulated with prolactin, cultured bovine mammary epithelial cellspolarize and organize into structures capable of secreting certain milkcomponents (Blatchford et al. 1999 Animal Cell Technology: Basic &Applied Aspects 10:141-145). In Blatchford et al, bovine MECs polarizedand formed mammospheres. Casein and butyrophilin were isolated from thecultures. However, the cells did not polarize in one uniform direction.Blatchford, et al. noted that the milk proteins were distributed inbetween the cells and dispersed throughout the mammospheres. Due to thelack of a uniform polarization orientation, Blatchford had to isolatethe secreted proteins from the culture media.

Furthermore, in vitro two-dimensional models, such as those used inBlatchford et al. provide a low surface area-to-volume ratio (lowdensity format). The surface area available for cell attachment limitsthe number of cells that can be grown

The only known attempt to culture mouse mammary epithelial cells in ahigh-density format, such as the hollow fiber bioreactor, failed toachieve compartmentalization necessary for the production and extractionof a cultured milk product (Sharfstein et al. 1992 Biotechnology andBioengineering 40:672-680). In Sharfstein et al., growth, long-termexpression of functional differentiation, and metabolism of COMMA-1D (animmortalized mouse mammary epithelial cell line) was examined in twodifferent systems: extended batch culture and hollow-fiber reactorculture. Using COMMA-1D seeded onto Costar Transwell® polycarbonatemembrane cell culture inserts, Sharfstein et al. created a confluentmonolayer capable of barrier formation and polarized metabolism betweenthe apical and basal side that maintained gradients of glucose andlactate. However, using a hollow-fiber bioreactor culture, Sharfstein etal. was unable to achieve separation of basal and apical compartments.Furthermore, it was not determined if nutrient uptake was polarized in ahollow-fiber culture (Sharfstein et al. 1992). Importantly, no priorwork has been able to culture mammary epithelial cells from humans orother nutritionally relevant species in a high-density,three-dimensional, compartmentalizing format.

Disclosed herein, in certain embodiments, are live cell constructs,methods of making the same, and methods of using the same for in vitroand/or ex vivo production of cultured milk product from cultured mammarycells.

This description is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof

Unless the context indicates otherwise, it is specifically intended thatthe various features described herein can be used in any combination.Moreover in some embodiments, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a complex comprises components A, B and C, itis specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, any feature or combination of features set forth herein can beexcluded or omitted.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of a compound or agent, dose, time, temperature, andthe like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, oreven ±0.1% of the specified amount.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The terminology used in the description herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-1UB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for production of recombinant and syntheticpolypeptides, antibodies or antigen-binding fragments thereof,manipulation of nucleic acid sequences, production of transformed cells,the construction of viral vector constructs, and transiently and stablytransfected packaging cells. Such techniques are known to those skilledin the art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, NY, 1989); F. M. Ausubel et al.Current Protocols In Molecular Biology (Green Publishing Associates,Inc. and John Wiley & Sons, Inc., New York).

As used herein, the transitional phrase “consisting essentially of” isto be interpreted as encompassing the recited materials or steps andthose that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

As used herein, the term “polypeptide” encompasses both peptides andproteins, and does not require any particular amino acid length ortertiary structure unless indicated otherwise.

The term “polarized” as used herein in reference to cells and/ormonolayers of cells refers to a spatial status of the cell wherein thereare two distinct surfaces of the cell, e.g., an apical surface and abasal surface, which may be different. In some embodiments, the distinctsurfaces of a polarized cell comprises different surface and/ortransmembrane receptors and/or other structures. In some embodiments,individual polarized cells in a continuous monolayer havesimilarly-oriented apical surfaces and basal surfaces. In someembodiments, individual polarized cells in a continuous monolayer havecommunicative structures between individual cells (e.g., tightjunctions) to allow cross communication between individual cells and tocreate separation (e.g., compartmentalization) of the apical compartmentand basal compartment.

As used herein, “apical surface” means the surface of a cell that facesan external environment or toward a cavity or chamber, for example thecavity of an internal organ. With respect to mammary epithelial cells,the apical surface is the surface from which the cultured milk productis secreted.

As used herein, “basal surface” means the surface of a cell that is incontact with a surface, e.g., the matrix of a bioreactor.

As used herein, “bioreactor” means a device or system that supports abiologically active environment that enables the production of a culturemilk product described herein from mammary cells described herein.

The term “lactogenic” as used herein refers to the ability to stimulateproduction and/or secretion of milk. A gene or protein (e.g., prolactin)may be lactogenic, as may any other natural and/or synthetic product. Insome embodiments, a lactogenic culture medium comprises prolactin,thereby stimulating production of milk by cells in contact with theculture medium.

As used herein, the term “food grade” refers to materials considerednon-toxic and safe for consumption (e.g., human and/or other animalconsumption), e.g., as regulated by standards set by the U.S. Food andDrug Administration.

In some embodiments, milk produced by the primary mammary epithelialcells (e.g., primary mammary epithelial cells from the isolated liveprimary mammary epithelial cells and/or the primary mammary epithelialcells from the mixed population of live primary mammary epithelialcells, mammary myoepithelial cells and/or mammary progenitor cells) orthe immortalized mammary epithelial cells is secreted through the apicalsurface of the cells into the apical compartment. In some embodiments, abasal compartment comprises a culture medium and the culture medium isin contact with the basal surface of the live primary mammary epithelialcells, the mixed population of live primary mammary epithelial cells,mammary myoepithelial cells and mammary progenitor cells, and/or theimmortalized mammary epithelial cells.

Live Cell Constructs

Disclosed herein, in certain embodiments, are live cell constructs forproducing milk in culture, the live cell constructs comprising acontinuous monolayer of live mammary cells selected from the groupconsisting of: (a) live primary mammary epithelial cells, (b) livemammary myoepithelial cells, (c) live mammary progenitor cells, and/or(d) live immortalized mammary epithelial cells.

In some embodiments, the mammary cells comprise milk-producing mammaryepithelial cells, contractile myoepithelial cells, and/or progenitorcells that can give rise to both mammary epithelial and mammarycontractile myoepithelial cells. Mammary epithelial cells are the onlycells that produce milk. In some embodiments, the mammary cells comprisemammary epithelial cells, primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells.

In some embodiments, the mammary cells are from breast tissue, uddertissue, and/or teat tissue of a mammal. In some embodiments, the mammarycells are from any mammal, e.g., a primate (e.g., chimpanzee, orangutan,gorilla, monkey (e.g., Old World, New World), lemur, human), a dog, acat, a rabbit, a mouse, a rat, a horse, a cow, a goat, a sheep, an ox(e.g., Bos spp.), a pig, a deer, a musk deer, a bovid, a whale, adolphin, a hippopotamus, an elephant, a rhinoceros, a giraffe, a zebra,a lion, a cheetah, a tiger, a panda, a red panda, and an otter. In someembodiments, the mammary cells are from an endangered species, e.g., anendangered mammal. In some embodiments, the mammary cells are from ahuman. In some embodiments, the mammary cells are from a bovid (e.g., acow).

In some embodiments, the continuous monolayer of live mammary cells isderived from breast milk-derived stem cells or breast stem cellsoriginating from tissue biopsy of the mammary gland. The epithelialcomponent of breast milk includes not only mature epithelial cells, butalso their precursors and stem cells in culture. A subpopulation ofbreast milk-derived stem cells displays very high multilineagepotential, resembling those typical for human embryonic stem cells(hESCs). Breast stem cells may also originate from tissue biopsy of themammary gland, and include terminally differentiated MECs. Both breastmilk-derived stem cells and breast stem cells originating from tissuebiopsy of the mammary gland are multi-potent cells that can give rise toMECs or myoepithelial cells.

In some embodiments, at least 50% of the mammary cells of the live cellsculture are polarized. In some embodiments, at least 55% of the mammarycells of the live cells culture are polarized. In some embodiments, atleast 60% of the mammary cells of the live cells culture are polarized.In some embodiments, at least 65% of the mammary cells of the live cellsculture are polarized. In some embodiments, at least 70% of the mammarycells of the live cells culture are polarized. In some embodiments, atleast 75% of the mammary cells of the live cells culture are polarized.In some embodiments, at least 80% of the mammary cells of the live cellsculture are polarized. In some embodiments, at least 85% of the mammarycells of the live cells culture are polarized. In some embodiments, atleast 90% of the mammary cells of the live cells culture are polarized.In some embodiments, at least 95% of the mammary cells of the live cellsculture are polarized. In some embodiments, at least 100% of the mammarycells of the live cells culture are polarized. In some embodiments,substantially all of the mammary cells of the live cell construct arepolarized (i.e., have an apical surface and a basal surface). In someembodiments, substantially all of the mammary cells of the live cellconstruct are polarized and substantially all of the polarized cells areoriented in the same direction. For example, in some embodiments,substantially all of the mammary cells have an apical surface and abasal surface, wherein the apical surface of substantially all of thecells is oriented in the same direction and the basal surface ofsubstantially all of the cells is oriented in the same direction.

In some embodiments, the monolayer of epithelial mammary cells has atleast 70% confluence over the scaffold. In some embodiments, themonolayer of mammary epithelial cells has at least about 75% confluenceover the scaffold. In some embodiments, the monolayer of epithelialmammary cells has at least about 80% confluence over the scaffold. Insome embodiments, the monolayer of epithelial mammary cells has at leastabout 85% confluence over the scaffold. In some embodiments, themonolayer of epithelial mammary cells has at least about 90% confluenceover the scaffold. In some embodiments, monolayer of epithelial mammarycells has at least about 95% confluence over the scaffold. In someembodiments, the monolayer of epithelial mammary cells has at leastabout 99% confluence over the scaffold. In some embodiments, themonolayer of epithelial mammary cells has 100% confluence over thescaffold.

Genetic Modifications to Mammary Cells

In some embodiments, the mammary cells comprise a constitutively activeprolactin receptor protein. In some embodiments, the mammary cellscomprise a constitutively active human prolactin receptor protein. Wherethe primary mammary epithelial cell or immortalized mammary epithelialcells comprise a constitutively active prolactin receptor, the culturemedium does not contain prolactin.

In some embodiments, the constitutively active human prolactin receptorprotein comprises a deletion of amino acids 9 through 187, wherein thenumbering is based on the reference amino acid sequence of a humanprolactin receptor identified as SEQ ID NO: 1.

SEQ ID NO: 1: Human prolactin receptor (GenBankaccession number AAD32032.1)MKENVASATVFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTDGGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPEKAAEWEIHFAGQQTEFKILSLHPGQKYLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDTTVWISVAVLSAVICLIIVWAVALKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGCQDFPPTSDYEDLLVEYLEVDDSEDQHLMSVHSKEHPSQGMKPTYLDPDTDSGRGSCDSPSLLSEKCEEPQANPSTFYDPEVIEKPENPETTHTWDPQCISMEGKIPYFHAGGSKCSTWPLPQPSQHNPRSSYHNITDVCELAVGPAGAPATLLNEAGKDALKSSQTIKSREEGKATQQREVESFHSETDQDTPWLLPQEKTPFGSAKPLDYVEIHKVNKDGALSLLPKQRENSGKPKKPGTPENNKEYAKVSGVMDNNILVLVPDPHAKNVACFEESAKEAPPSLEQNQAEKALANFTATSSKCRLQLGGLDYLDPACFTHSFH

In some embodiments, the constitutively active human prolactin receptorprotein comprising a deletion of the following amino acids:

VFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTDGGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPEKAA (e.g., amino acid positions 10through 178 of SEQ ID NO: 1).

In some embodiments, the mammary cells comprise a loss of functionmutation introduced into a circadian related gene PER2. In someembodiments, the loss of function mutation introduced into a circadianrelated gene PER2 promotes increased synthesis of cultured milkcomponents. In some embodiments, the loss of function mutation in thePER2 gene comprises an 87-amino acid deletion from position 348 to 434in PER2, wherein the numbering is based on the reference amino acidseauence of a human PER2 identified as SEO ID NO:2.

SEQ ID NO: 2: Human Period circadian protein homo-log 2 (GenBank accession number NM_022817)MNGYAEFPPSPSNPTKEPVEPQPSQVPLQEDVDMSSGSSGHETNENCSTGRDSQGSDCDDSGKJELGMLVEPPDARQSPDTFSLMMAKSEHNPSTSGCSSDQSSKVDTHKEL1KTLKELKVHLPADKKAKGKASTLATLKYALRSVKQVKANEEYYQLLMSSEGHPCGADVPSYTVEEMESVTSEHIVKNADMFAVAVSLVSGKILYISDQVASIFHCKRDAFSDAKFVEFLAPHDVGVFHSFTSPYKLPLWSMCSGADSFTQECMEEKSFFCRVSVRKSHENEIRYHPFRMTPYLVKVRDQQGAESQLCCLLLAERVHSGYEAPRIPPEKRIFTTTHTPNCLFQDVDERAVPLLGYLPQDLIETPVLVQLHPSDRPLMLAIHKKILQSGGQPFDYSPIRFRARNGEYITLDTSWSSFINPWSRKISFIIGRHKVRVGPLNEDVFAAHPCTEEKALHPSIQELTEQIHRLLLQPVPHSGSSGYGSLGSNGSHEHLMSQTSSSDSNGHEDSRRRRAEICKNGNKTKNRSHYSHESGEQKKKSVTEMQTNPPAEKKAVPAMEKDSLGVSFPEELACKNQPTCSYQQISCLDSVIRYLESCNEAATLKRKCEFPANVPALRSSDKRKATVSPGPHAGEAEPPSRVNSRTGVGTHLTSLALPGKAESVASLTSQCSYSSTIVHVGDKKPQPELEMVEDAASGPESLDCLAGPALACGLSQEKEPFKKLGLTKEVLAAHTQKEEQSFLQKFKEIRKLSIFQSHCHYYLQERSKGQPSERTAPGLRNTSGIDSPWKKTGKNRKLKSKRVKPRDSSESTGSGGPVSARPPLVGLNATAWSPSDTSQSSCPAVPFPAPVPAAYSLPVFPAPGTVAAPPAPPHASFTVPAVPVDLQHQFAVQPPPFPAPLAPVMAFMLPSYSFPSGTPNLPQAFFPSQPQFPSHPTLTSEMASASQPEFPEGGTGAMGTTGATETAAVGADCKPGTSRDQQPKAPLTRDEPSDTQNSDALSTSSGLLNLLLNEDLCSASGSAASESLGSGSLGCDASPSGAGSSDTSHTSKYFGSIDSSENNHKAKMNTGMEESEHFIKCVLQDPIWLLMADADSSVMMTYQLPSRNLEAVLKEDREKLKLLQKLQPRFTESQKQELREVHQWMQTGGLPAAIDVAECVYCENKEKGNICIPYEEDIPSLGLSEVSDTKEDENGSPLNH RIEEQT

In some embodiments, the loss of function mutation introduced into PER2comprises a deletion of the following amino acids:

CLFQDVDERAVPLLGYLPQDLIETPVLVQLHPSDRPLMLAIHKKILQSGGQPFDYSPIRFRARNGEYITLDTSWSSFINPWSRKISFIIGRHKV(e.g., amino acid positions 341 through 434 of SEQ ID NO: 2).

In some embodiments, the mammary cells comprise a polynucleotideencoding a prolactin receptor comprising a modified intracellularsignaling domain. In some embodiments, the loss of function mutationintroduced into a circadian related gene PER2 promotes increasedsynthesis of individual cultured milk components. In some embodiments,the prolactin receptor comprises a truncation wherein position 154 ofexon 10 has been spliced to the 3′ sequence of exon 11. In someembodiments, the prolactin receptor comprises a sequence according toSEQ ID NO: 3.

SEQ ID NO: 3: Human isoform 4 of Prolactin recep-tor (GenBank accession number AF416619; Trott etal. 2003 J. Mol. Endocrinol 3Q(1): 31-47)MKENVASATVFTLLLFLNTCLLNGQLPPGKPEIFKCRSPNKETFTCWWRPGTDGGLPTNYSLTYHREGETLMHECPDYITGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYVDVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTGWFTLLYEIRLKPEKAAEWEIHFAGQQTEFKILSLHPGQKYLVQVRCKPDHGYWSAWSPATFIQIPSDFTMNDTTVWISVAVLSAVICLIIVWAVALKGYSMVTCIFPPVPGPKIKGFDAHLLEKGKSEELLSALGCQDFPPTSDYEDLLVEYLEVDDSEDQHLMSVHSKEHPSQGDPLMLGASHYKNLKSYRPRKISSQGRLAVFTKATLTTVQ

In some embodiments, the mammary cells comprise a polynucleotideencoding a modified (e.g., recombinant) effector of a prolactin protein.In some embodiments, the modified effector of the prolactin proteincomprises a janus kinase-2 (JAK2) tyrosine kinase domain. In someembodiments, the modified effector comprises a JAK2 tyrosine kinasedomain fused to a signal transducer and activator of transcription-5(STAT5) tyrosine kinase domain (e.g., a polynucleotide encoding a JAK2tyrosine kinase domain linked to the 3′ end of a polynucleotide encodingthe STAT5 tyrosine kinase domain). In some embodiments, the modifiedeffector of a prolactin protein promotes increased synthesis ofindividual cultured milk components. In some embodiments, the modifiedeffector has a sequence according to SEQ ID NO: 4. Bolded amino acidscorrespond to the JAK2 kinase domain of amino acid positions 757 through1129 of a reference human JAK2 amino acid sequence.

SEQ ID NO: 4. STA5A Human signal transducer andactivator of transcription 5A fused at 3′ endto amino acids 757-1129 of JAK2 human tyrosine- protein kinaseMAGWIQAQQL QGDALRQMQV LYGQHFPIEV RHYLAQWIESQPWDAIDLDN PQDRAQATQL LEGLVQELQK KAEHQVGEDGFLLKIKLGHY ATQLQKTYDR CPLELVRCIR HILYNEQRLVREANNCSSPA GILVDAMSQK HLQINQTFEE LRLVTQDTENELKKLQQTQE YFIIQYQESL RIQAQFAQLA QLSPQERLSRETALQQKQVS LEAWLQREAQ TLQQYRVELA EKHQKTLQLLRKQQTIILDD ELIQWKRRQQ LAGNGGPPEG SLDVLQSWCEKLAEIIWQNR QQIRRAEHLC QQLPIPGPVE EMLAEVNATITDIISALVTS TFIIEKQPPQ VLKTQTKFAA TVRLLVGGKLNVHMNPPQVK ATIISEQQAK SLLKNENTRN ECSGEILNNCCVMEYHQATG TLSAHFRNMS LKRIKRADRR GAESVTEEKFTVLFESQFSV GSNELVFQVK TLSLPWVIV HGSQDHNATATVLWDNAFAE PGRVPFAVPD KVLWPQLCEA LNMKFKAEVQSNRGLTKENL VFLAQKLFNN SSSHLEDYSG LSVSWSQFNRENLPGWNYTF WQWFDGVMEV LKKHHKPHWN DGAILGFVNKQQAHDLLINK PDGTFLLRFS DSEIGGITIA WKFDSPERNLWNLKPFTTRD FSIRSLADRL GDLSYLIYVF PDRPKDEVFSKYYTPVLAKA VDGYVKPQIK QWPEFVNAS ADAGGSSATYMDQAPSPAVC PQAPYNMYPQ NPDHVLDQDG EFDLDETMDVARHVEELLRR PMDSLDSRLS PPAGLFTSAR GSLSLDSQRKLQFYEDRH QLPAPKWAEL ANLINNCMDY EPDFRPSFRAIIRDLNSLFT PDYELLTEND MLPNMRIGAL GFSGAFEDRDPTQFEERHLK FLQQLGKGNF GSVEMCRYDP LQDNTGEWAVKKLQHSTEE HLRDFEREIE ILKSLQHDNI VKYKGVCYSAGRRNLKLIME YLPYGSLRDY LQKHKERIDH IKLLQYTSQICKGMEYLGTK RYIHRDLATR NILVENENRV KIGDFGLTKVLPQDKEYYKV KEPGESPIFW YAPESLTESK FSVASDVWSFGWLYELFTY IEKSKSPPAE FMRMIGNDKQ GQMIVFHLIELLKNNGRLPR PDGCPDEIYM IMTECWNNNV NQRPSFRDLA LRVDQIRDN.

In some embodiments, the mammary cells are immortalized. In someembodiments, the mammary cells comprise one or more nucleic acidsencoding human telomerase reverse transcriptase (hTERT) or simian virus40 (SV40). In some embodiments, the mammary cells comprise a smallhairpin RNA (shRNA) to p16 (Inhibitor of Cyclin-Dependent Kinase 4)(p16(INK4)) and Master Regulator of Cell Cycle Entry and ProliferativeMetabolism (c-MYC).

In some embodiments, the method comprises introducing into the cell: (a)a polynucleotide encoding a prolactin receptor comprising a modifiedintracellular signaling domain, optionally wherein the prolactinreceptor comprises a truncation wherein position 154 of exon 10 has beenspliced to the 3′ sequence of exon 11; (b) a polynucleotide encoding achimeric prolactin receptor that binds to a ligand, which is capable ofactivating milk synthesis in the absence of prolactin; (c) apolynucleotide encoding a constitutively or conditionally activeprolactin receptor protein, optionally wherein the polynucleotideencodes a constitutively active human prolactin receptor proteincomprising a deletion of amino acids 9 through 187 (e.g., a deletion ofamino acids 9 through 187, wherein the numbering is based on thereference amino acid sequence of a human prolactin receptor identifiedas SEQ ID NO: 1); (d) a polynucleotide encoding a modified (e.g.,recombinant) effector of a prolactin protein comprising (i) a januskinase-2 (JAK2) tyrosine kinase domain, optionally wherein the JAK2tyrosine kinase domain is fused to a signal transducer and activator oftranscription-5 (STATS) tyrosine kinase domain (e.g., a polynucleotideencoding a JAK2 tyrosine kinase domain linked to the 3′ end of apolynucleotide encoding the STATS tyrosine kinase domain); and/or (ii) aprolactin receptor intracellular domain fused to a JAK2 tyrosine kinasedomain; (e) a loss of function mutation into a circadian related genePER2 (period circadian protein homolog 2); and/or (f) a polynucleotideencoding one or more glucose transporter genes GLUT1 and/or GLUT12,thereby increasing the rate of nutrient uptake at the basal surface ofthe monolayer.

Scaffolds

In some embodiments, the live cell construct further comprises ascaffold having a top surface/exterior surface and a bottomsurface/interior surface. In some embodiments, the scaffold is a2-dimensional surface or a 3-dimensional surface (e.g., a 3-dimensionalmicropatterned surface, and/or as a cylindrical structure that isassembled into bundles). A non-limiting example of a 2-dimensionalsurface scaffold is a Transwell® filter. In some embodiments, thescaffold is a 3-dimensional surface. Non-limiting examples of a3-dimensional micropatterned surface include a microstructuredbioreactor, a decellularized tissue (e.g., a decellularized mammarygland or decellularized plant tissue), micropatterned scaffoldsfabricated through casting or three-dimensional printing with biologicalor biocompatible materials, textured surface. In some embodiments, thescaffold is produced by electrospinning cellulose nanofibers and/or acylindrical structure that can be assembled into bundles (e.g., a hollowfiber bioreactor). In some embodiments, the scaffold is porous. In someembodiments, the scaffold is a 3D scaffold. In some embodiments, the3-dimensional scaffold is any structure which has an enclosed hollowinterior/central cavity. In some embodiments, the three dimensionalscaffold joins with one or more surfaces to form an enclosed interiorchamber/basal compartment. For example, the scaffold can join with oneor more walls of a bioreactor to form the interior chamber/basalcompartment. In some embodiments, the scaffold is a hollow fiberbioreactor. In some embodiments, the 3D scaffold is a tube in which thecentral cavity is defined by the interior surface of the scaffold. Insome embodiments, the 3D scaffold is a hollow sphere in which thecentral cavity is defined by the interior surface of the scaffold.

For in vitro culture methods for studies of intestinal absorption,2-dimensional surface scaffold such as Transwells® have long been usedas the standard as they provide both apical and basolateral spaces tosimulate the gut-blood-barrier and enable both active and passivetransport of drugs and nutrients. However, cells seeded onto flatsupports exhibit markedly different phenotypes to cells in vivo, partlydue to the poor representation of the 3-D extracellularmicroenvironments.

A 3-dimensional scaffold allows mammary cells (e.g., MECs) to grow orinteract with their surroundings in all three dimensions. Unlike 2Denvironments, a 3D cell culture allows cells in vitro to grow in alldirections, approximating the in vivo mammary environment. Further, the3D scaffold allows for a larger surface area for culture of the cellsand for metabolite and gas exchange, plus it enables necessarycompartmentalization—enabling the cultured milk product to be secretedinto one compartment, while the cell culture media is contacted with themammary cells in another compartment. To date, a confluent monolayerwith polarized separation of basal and apical cell surfaces usingmammary epithelial cell on a 3D surface has not been achieved(Sharfstein et al. 1992).

In some embodiments, the scaffold is porous. In some embodiments, thescaffold is permeable to the cell media, allowing the cell media tocontact the cells of the cell monolayer. In some embodiments, thescaffold is transversed by at least one pore that allows the cell mediato contact the basal surface of the cells of the cell monolayer.

In some embodiments, the top surface/exterior surface of the scaffold iscoated with a matrix material. In some embodiments, the matrix is madeup of one or more extracellular matrix proteins. Non-limiting examplesof extracellular matrix proteins include collagen, laminin, entactin,tenascin, and/or fibronectin. In some embodiments, the scaffoldcomprises a natural polymer, a biocompatible synthetic polymer, asynthetic peptide, and/or a composite derived from any combinationthereof. In some embodiments, a natural polymer useful with thisinvention includes, but is not limited to, collagen, chitosan,cellulose, agarose, alginate, gelatin, elastin, heparan sulfate,chondroitin sulfate, keratan sulfate, and/or hyaluronic acid. In someembodiments, a biocompatible synthetic polymer useful with thisinvention includes, but is not limited to, cellulose, polysulfone,polyvinylidene fluoride, polyethylene co-vinyl acetate, polyvinylalcohol, sodium polyacrylate, an acrylate polymer, and/or polyethyleneglycol. In some embodiments, the top of the scaffold is coated withlaminin and collagen.

In some embodiments, the matrix material is porous. In some embodiments,the matrix material is permeable to the cell media, allowing the cellmedia to contact the cells of the cell monolayer. In some embodiments,the matrix material is transversed by at least one pore that allows thecell media to contact the basal surface of the cells of the cellmonolayer.

In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 0.1 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 0.2 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 0.3 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 0.4 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about0.5 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 0.6 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 0.7 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 0.8 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 0.9 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about1.0 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 1.1 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 1.2 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 1.3 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 1.4 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about1.5 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 1.6 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 1.7 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 1.8 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 1.9 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about2.0 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 2.1 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 2.2 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 2.2 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 2.3 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about2.4 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 2.5 μm. In some embodiments, the pore size ofthe scaffold and/or matrix material is at least about 2.6 μm. In someembodiments, the pore size of the scaffold and/or matrix material is atleast about 2.7 μm. In some embodiments, the pore size of the scaffoldand/or matrix material is at least about 2.8 μm. In some embodiments,the pore size of the scaffold and/or matrix material is at least about2.9 μm. In some embodiments, the pore size of the scaffold and/or matrixmaterial is at least about 3.0 μm.

In some embodiments, the live cell construct comprises a scaffold havinga top surface/exterior surface and a bottom surface/interior surface;and a continuous monolayer of (a) live primary mammary epithelial cells,(b) a mixed population of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, and/or (c) liveimmortalized mammary epithelial cells on the top surface of thescaffold, the continuous monolayer of (a) live primary mammaryepithelial cells, (b) mixed population of live primary mammaryepithelial cells mammary myoepithelial cells and mammary progenitorcells, and/or (c) immortalized mammary epithelial cells having an apicalsurface and a basal surface (e.g., the cells form a polarized andconfluent cell monolayer), wherein the construct comprises an apicalcompartment above and adjacent to the apical surface of the continuousmonolayer of the (a) live primary mammary epithelial cells, the (b)mixed population of live primary mammary epithelial cells, mammarymyoepithelial cell and mammary progenitor cells, and/or the (c)immortalized mammary epithelial cells and a basal compartment below andadjacent to the bottom surface of the scaffold.

Bioreactor

Disclosed herein, in certain embodiments, are bioreactors, comprising:(a) an apical compartment comprising a cultured milk product; and (b) atleast one live cell construct comprising: (i) a three-dimensionalscaffold having an exterior surface, an interior surface defining aninterior cavity/basal chamber, and a plurality of pores extending fromthe interior surface to the exterior surface; (ii) a matrix materialdisposed on the exterior surface of the three-dimensional scaffold;(iii) a culture media disposed within the interior cavity/basal chamberand in fluidic contact with the internal surface; and (iv) an at least70% confluent monolayer of polarized mammary cells disposed on thematrix material, wherein the mammary cells are selected from the groupconsisting of: live primary mammary epithelial cells, live mammarymyoepithelial cells, live mammary progenitor cells, live immortalizedmammary epithelial cells, live immortalized mammary myoepithelial cells,and live immortalized mammary progenitor cells; wherein the apicalsurface of the mammary cells is in fluidic contact with the apicalcompartment.

In some embodiments, the bioreactor is an enclosed bioreactor. In someembodiments, the apical chamber is substantially isolated from theinterior cavity/basal compartment.

A hollow fiber bioreactor is an exemplary bioreactor for use with themethods disclosed here. The hollow fiber bioreactor is a high-density,continuous perfusion culture system that closely approximates theenvironment in which cells grow in vivo. It consists of thousands ofsemi-permeable 3D scaffolds (i.e., hollow fibers) in a parallel arraywithin a cartridge shell fitted with inlet and outlet ports. These fiberbundles are potted or sealed at each end so that any liquid entering theends of the cartridge will necessarily flow through the interior of thefibers. Cells are generally seeded outside the fibers within thecartridge in the extra capillary space (ECS).

Three fundamental characteristics differentiate hollow fiber cellculture from other methods: (1) cells are bound to a porous matrix muchas they are in vivo, not a plastic dish, microcarrier or otherimpermeable support, (2) the molecular weight cut off of the supportmatrix can be controlled, and (3) extremely high surface area to volumeratio (150 cm² or more per mL) which provides a large area formetabolite and gas exchange for efficient growth of host cells.

The bioreactor structure provides a fiber matrix that allows permeationof nutrients, gases and other basic media components, as well as cellwaste products, but not cells, where the cells can be amplified. Hollowfiber bioreactor technology has been used to obtain high density cellamplification by utilizing hollow fibers to create a semi-permeablebarrier between the cell growth chamber and the medium flow. Since thesurface area provided by this design is large, using this fiber as aculture substrate allows the production of large numbers of cells. Cellsgrowing in the 3-dimensional environment within the bioreactor arebathed in fresh medium as it perfuses through the hollow fibers.

To replicate the topography of the intestine, Costello et al. developeda 3-D printed bioreactor that can both contain porous villus scaffoldsvia micromolding (Costello et al. 2017 Scientific Reports 7(12515):1-10). This geometrically complex molded scaffold provided separation ofthe apical and basolateral spaces in a manner in which fluid flowexposes intestinal epithelial cells to physiologically relevant shearstresses (Costello et al. 2017). Similarly, a long-term culture in vitroculture in a simulated gut-like environment was created by Morada et al.using a hollow fiber bioreactor which allowed for two controlledseparate environments (biphasic) to provide host cells with oxygen andnutrients from the basal layer, while allowing a low oxygen nutrientrich environment to be developed on the apical surface (Morada et al.2016 International Journal for Parasitology 26: 21-29).

In configuring the hollow fiber bioreactor, there are designconsiderations and parameters that can be varied depending upon thegoals associated with expansion of the cells. One such designconsideration is the size of the pores in the fiber wall. This isgenerally designed to allow the passage of nutrients to the cells, carryaway waste, provide desired products to the cells (such as growthfactors), to remove desired products from the cells, and exclude certainfactors that may be present from reaching the cells. Accordingly, thepore size of the fiber walls can be varied to modify which componentswill pass through the walls. For example, pore size can allow thepassage of large proteinaceous molecules, including growth factors,including, but not limited to, epidermal growth factor andplatelet-derived growth factor. The person of ordinary skill in the artwould understand how to vary the pore size depending upon the componentsthat it is desirable to pass through the fiber walls to reach the cellsor to carry material from the cells.

In some embodiments, the pore size is about 0.2 μm. In some embodiments,the pore size is about 0.1. In some embodiments, the pore size is about0.2 μm. In some embodiments, the pore size is about 0.3 μm. In someembodiments, the pore size is about 0.4 μm. In some embodiments, thepore size is about 0.5 μm. In some embodiments, the pore size is about0.6 μm. In some embodiments, the pore size is about 0.7 μm. In someembodiments, the pore size is about 0.8 μm. In some embodiments, thepore size is about 0.9 μm. In some embodiments, the pore size is about1.0 μm. In some embodiments, the pore size is about 1.1 μm. In someembodiments, the pore size is about 1.2 μm. In some embodiments, thepore size is about 1.3 μm. In some embodiments, the pore size is about1.4 μm. In some embodiments, the pore size is about 1.5 μm. In someembodiments, the pore size is about 1.6 μm. In some embodiments, thepore size is about 1.7 μm. In some embodiments, the pore size is about1.8 μm. In some embodiments, the pore size is about 1.9 μm. In someembodiments, the pore size is about 2.0 μm. In some embodiments, thepore size is about 2.1 μm. In some embodiments, the pore size is about2.2 μm. In some embodiments, the pore size is about 2.2 μm. In someembodiments, the pore size is about 2.3 μm. In some embodiments, thepore size is about 2.4 μm. In some embodiments, the pore size is about2.5 μm. In some embodiments, the pore size is about 2.6 μm. In someembodiments, the pore size is about 2.7 μm. In some embodiments, thepore size is about 2.8 μm. In some embodiments, the pore size is about2.9 μm. In some embodiments, the pore size is about 3.0 μm.

Methods of Making Live Cell Constructs

Disclosed herein, in certain embodiments, are methods of making a livecell construct for producing a cultured milk product. In someembodiments, the method comprises (a) isolating primary mammaryepithelial cells, myoepithelial cells and/or mammary progenitor cellsfrom mammary explants from mammary tissue (e.g., breast, udder, teattissue), biopsy sample, or raw breastmilk, to produce isolated mammaryepithelial cells, myoepithelial cells and/or mammary progenitor cells;(b) culturing the isolated primary mammary epithelial cells,myoepithelial cells and/or mammary progenitor cells to produce a mixedpopulation of primary mammary epithelial cells, mammary myoepithelialcells and mammary progenitor cells; (c) cultivating the mixed populationof (b) on a scaffold having an upper surface and lower surface, toproduce a polarized, monolayer of primary mammary epithelial cells,myoepithelial cells and mammary progenitor cells of the mixed populationon the upper surface of the scaffold, wherein the polarized monolayercomprises an apical surface and a basal surface, thereby producing alive cell construct for producing the cultured milk product.

In some embodiments, the method comprises: a) isolating primary mammaryepithelial cells, myoepithelial cells, and/or mammary progenitor cellsfrom mammary explants from mammary tissue (e.g., breast, udder, teattissue), biopsy sample, or raw breastmilk, to produce isolated mammaryepithelial cells, myoepithelial cells, and/or mammary progenitor cells;(b) culturing the isolated primary mammary epithelial cells,myoepithelial cells, and/or mammary progenitor cells to produce a mixedpopulation of primary mammary epithelial cells, mammary myoepithelialcells and mammary progenitor cells; (c) sorting the mixed population ofprimary mammary epithelial cells, myoepithelial cells, and/or mammaryprogenitor cells (e.g., selecting the primary mammary epithelial cells)to produce a population of primary mammary epithelial cells; and (d)cultivating the population of primary mammary epithelial on a scaffoldhaving an upper surface and lower surface, to produce a polarizedmonolayer of primary mammary epithelial cells on the upper surface ofthe scaffold, wherein the polarized monolayer comprises an apicalsurface and a basal surface, thereby producing a live cell construct forproducing the cultured milk product.

In some embodiments, the method comprises (a) culturing immortalizedmammary epithelial cells to produce increased numbers of immortalizedmammary epithelial cells; (b) cultivating the immortalized mammaryepithelial cells of (a) on a scaffold, the scaffold having an uppersurface and lower surface, to produce a polarized monolayer ofimmortalized mammary epithelial cells on the upper surface of thescaffold, wherein the polarized monolayer comprises an apical surfaceand a basal surface, thereby producing a live cell construct forproducing the cultured milk product.

In some embodiments, the culturing and/or cultivating of the mammarycells for the live cell construct is carried out at a temperature ofabout 35° C. to about 39° C. (e.g., a temperature of about 35° C., 35.5°C., 36° C., 36.5° C., 37° C., 37.5° C., 38° C., 38.5° C. or about 39°C., or any value or range therein, e.g., about 35° C. to about 38° C.,about 36° C. to about 39° C., about 36.5° C. to about 39° C., about36.5° C. to about 37.5° C., or about 36.5° C. to about 38° C.). In someembodiments, the culturing and/or cultivating is carried out at atemperature of about 37° C.

In some embodiments, the culturing and/or cultivating of the mammarycells for the live cell construct is carried out at an atmosphericconcentration of CO₂ of about 4% to about 6%, e.g., an atmosphericconcentration of CO₂ of about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%,5.75%, or 6% or any value or range therein, e.g., about 4% to about5.5%, about 4.5% to about 6%, about 4.5% to about 5.5%, or about 5% toabout 6%). In some embodiments, the culturing and/or cultivating iscarried out at an atmospheric concentration of CO₂ of about 5%.

In some embodiments, the culturing and/or cultivating of the mammarycells for the live cell construct comprises culturing and/or cultivatingin a culture medium that is exchanged about every day to about every 10days (e.g., every 1 day, every 2 days, every 3 days, every 4 days, every5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10days, or any value or range therein, e.g., about every day to every 3days, about every 3 days to every 10 days, about every 2 days to every 5days). In some embodiments, the culturing and/or cultivating furthercomprises culturing in a culture medium that is exchanged about everyday to about every few hours to about every 10 days, e.g., about every1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, or 24 hours to about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10days or any value or range therein. For example, in some embodiments,the culturing and/or cultivating further comprises culturing and/orcultivating in a culture medium that is exchanged about every 12 hoursto about every 10 days, about every 10 hours to about every 5 days, orabout every 5 hours to about every 3 days.

In some embodiments, the live cell construct is stored in a freezer orin liquid nitrogen. The storage temperature depends on the desiredstorage length. For example, freezer temperature (e.g., storage at atemperature of about 0° C. to about −80° C. or less, e.g., about 0° C.,−10° C., −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C.,−90° C., −100° C. or any value or range therein) may be used if thecells are to be used within 6 months (e.g., within 1, 2, 3, 4, 5, or 6months). For example, liquid nitrogen may be used (e.g., storage at atemperature of -100° C. or less (e.g., about −100° C., −110° C., −120°C., −130, −140, −150, −160, −170, −180, −190° C., −200° C., or less) forlonger term storage (e.g., storage of 6 months or longer, e.g., 6, 7, 8,9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6 or more years).

In some embodiments, the mammary cells are isolated and sorted viafluorescence-activated cell sorting, magnetic-activated cell sorting,and/or microfluidic cell sorting.

Basal Culture Media and Lactogenic Media

In some embodiments, the culture medium comprises a carbon source, achemical buffering system, one or more essential amino acids, one ormore vitamins and/or cofactors, and one or more inorganic salts. In someembodiments, the carbon source, chemical buffering system, one or moreessential amino acids, one or more vitamins and/or cofactors, and/or oneor more inorganic salts are food grade.

In some embodiments, the culture medium is lactogenic culture medium. Insome embodiments, the culture medium further comprises prolactin (e.g.,mammalian prolactin, e.g., human prolactin), linoleic and alpha-linoleicacid, estrogen and/or progesterone. For example, in some embodiments,the culture medium comprises prolactin (or prolactin is added) in anamount from about 20 ng/mL to about 200 ng/L of culture medium, e.g.,about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 ng/mL or any value or range therein. In someembodiments, the culture medium comprises prolactin (or prolactin isadded) in an amount from about 20 ng/mL to about 195 ng/mL, about 50ng/mL to about 150 ng/mL, about 25 ng/mL to about 175 ng/mL, about 45ng/mL to about 200 ng/mL, or about 75 ng/mL to about 190 ng/mL ofculture medium. In some embodiments, the culture medium furthercomprises other factors to improve efficiency, including, but notlimited to, insulin, an epidermal growth factor, and/or ahydrocortisone.

In some embodiments, the culture medium comprises a carbon source in anamount from about 1 g/L to about 15 g/L of culture medium (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g/L or any value orrange therein), or about 1, 2, 3, 4, 5 or 6 g/L to about 7, 8, 9, or 10,11, 12, 13, 14 or 15 g/L of the culture medium. Non-limiting examples ofa carbon source include glucose and/or pyruvate. For example, in someembodiments, the culture medium comprises glucose in an amount fromabout Ig/L to about 12 g/L of culture medium, e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 g/L or any value or range therein. In someembodiments, the culture medium comprises glucose in an amount fromabout 1 g/L to about 6 g/L, about 4 g/L to about 12 g/L, about 2.5 g/Lto about 10.5 g/L, about 1.5 g/L to about 11.5 g/L, or about 2 g/L toabout 10 g/L of culture medium. In some embodiments, the culture mediumcomprises glucose in an amount from about 1, 2, 3, or 4 g/L to about 5,6, 7, 8, 9, 10, 11, or 12 g/L or about 1, 2, 3, 4, 5, or 6 g/L to about7, 8, 9, 10, 11, or 12 g/L. In some embodiments, the culture mediumcomprises pyruvate in an amount from about 5 g/L to about 15 g/L ofculture medium, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g/Lor any value or range therein. In some embodiments, the culture mediumcomprises pyruvate in an amount from about 5 g/L to about 14.5 g/L,about 10 g/L to about 15 g/L, about 7.5 g/L to about 10.5 g/L, about 5.5g/L to about 14.5 g/L, or about 8 g/L to about 10 g/L of culture medium.In some embodiments, the culture medium comprises pyruvate in an amountfrom about 5, 6, 7, or 8 g/L to about 9, 10, 11, 12, 13, 14 or 15 g/L orabout 5, 6, 7, 8, 9, or 10 g/L to about 11, 12, 13, 14 or 15 g/L.

In some embodiments, the culture medium comprises a chemical bufferingsystem in an amount from about 1 g/L to about 4 g/L (e.g., about 1, 1.5,2, 2.5, 3, 3.5, or 4 g/L or any value or range therein) of culturemedium or about 10 mM to about 25 mM (e.g., about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM or any value or rangetherein). In some embodiments, the chemical buffering system includes,but is not limited to, sodium bicarbonate and/or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). For example,in some embodiments, the culture medium comprises sodium bicarbonate inan amount from about 1 g/L to about 4 g/L of culture medium, e.g., about1, 1.5, 2, 2.5, 3, 3.5, or 4 g/L or any value or range therein. In someembodiments, the culture medium comprises sodium bicarbonate in anamount from about 1 g/L to about 3.75 g/L, about 1.25 g/L to about 4g/L, about 2.5 g/L to about 3 g/L, about 1.5 g/L to about 4 g/L, orabout 2 g/L to about 3.5 g/L of culture medium. In some embodiments, theculture medium comprises HEPES in an amount from about 10 mM to about 25mM, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 mM or any value or range therein. In some embodiments, theculture medium comprises HEPES in an amount from about 11 mM to about 25mM, about 10 mM to about 20 mM, about 12.5 mM to about 22.5 mM, about 15mM to about 20.75 mM, or about 10 mM to about 20 mM.

In some embodiments, the culture medium comprises one or more essentialamino acids in an amount from about 0.5 mM to about 5 mM (e.g., about0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mM or any value or rangetherein) or about 0.5, 1, 1.5, 2 mM to about 2.5, 3, 3.5, 4, 4.5, or 5mM. In some embodiments, the one or more essential amino acids ishistidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, valine, and/or arginine. For example, in someembodiments, the culture medium comprises arginine in an amount fromabout 0.5 mM to about 5 mM, e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, or 5 mM or any value or range therein. In some embodiments, theculture medium comprises an essential amino acids in an amount fromabout 0.5 mM to about 4.75 mM, about 2 mM to about 3.5 mM, about 0.5 mMto about 3.5 mM, about 1 mM to about 5 mM, or about 3.5 mM to about 5mM.

In some embodiments, the culture medium comprises one or more vitaminsand/or cofactors in an amount from about 0.01 μM to about 50 μM (e.g.,about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6, 7,8,9, 10, 12.5, 15,17.5,20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075, or50 μM or any value or range therein) or about 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or0.9 μM to about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6 μM or about 0.02, 0.025, 0.05, 0.075, 1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM to about 12.5, 15, 17.5, 20, 25, 30,35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075, or 50 μM. In someembodiments, one or more vitamins and/or cofactors include, but are notlimited to, thiamine and/or riboflavin. For example, in someembodiments, the culture medium comprises thiamine in an amount fromabout 0.025 μM to about 50 μM, e.g., about 0.025, 0.05, 0.075, 1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 46,47, 48, 49, 49.025, 49.05, 49.075, or 50 μM or any value or rangetherein. In some embodiments, the culture medium comprises thiamine inan amount from about 0.025 μM to about 45.075 μM, about 1 μM to about 40μM, about 5 μM to about 35.075 μM, about 10 μM to about 50 μM, or about0.05 μM to about 45.5 μM. In some embodiments, the culture mediumcomprises riboflavin in an amount from about 0.01 μM to about 3 μM,e.g., about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μMor any value or range therein. In some embodiments, the culture mediumcomprises riboflavin in an amount from about 0.01 μM to about 2.05 μM,about 1 μM to about 2.95 μM, about 0.05 μM to about 3 μM, about 0.08 μMto about 1.55 μM, or about 0.05 μM to about 2.9 μM.

In some embodiments, the culture medium comprises one or more inorganicsalts in an amount from about 100 mg/L to about 150 mg/L of culturemedium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or150 mg/L or any value or range therein) or about 100 mg/L to about 150mg/L of culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130,135, 140, 145, or 150 mg/L or any value or range therein). In someembodiments, one or more inorganic salts include, but are not limitedto, calcium and/or magnesium. For example, in some embodiments, theculture medium comprises calcium in an amount from about 100 mg/L toabout 150 mg/L of culture medium, e.g., about 100, 105, 110, 115, 120,125, 130, 135, 140, 145, or 150 mg/L or any value or range therein. Insome embodiments, the culture medium comprises arginine in an amountfrom about 100 mg/L to about 125 mg/L, about 105 mg/L to about 150 mg/L,about 120 mg/L to about 130 mg/L, or about 100 mg/L to about 145 mg/L ofculture medium. In some embodiments, the culture medium comprisesmagnesium in an amount from about 0.01 mM to about 1 mM, e.g., about0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98,0.99, or 1 mM or any value or range therein. In some embodiments, theculture medium comprises magnesium in an amount from about 0.05 mM toabout 1 mM, about 0.01 mM to about 0.78 mM, about 0.5 mM to about 1 mM,about 0.03 mM to about 0.75 mM, or about 0.25 mM to about 0.95 mM.

In some embodiments, the culture medium comprises a carbon source in anamount from about 1 g/L to about 15 g/L of culture medium (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g/L or any value orrange therein), or about 1, 2, 3, 4, 5 or 6 g/L to about 7, 8, 9, or 10,11, 12, 13, 14 or 15 g/L of the culture medium. In some embodiments, thecarbon source includes, but is not limited to, glucose and/or pyruvate.For example, in some embodiments, the culture medium comprises glucosein an amount from about 1 g/L to about 12 g/L of culture medium, e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 g/L or any value or rangetherein. In some embodiments, the culture medium comprises glucose in anamount from about 1 g/L to about 6 g/L, about 4 g/L to about 12 g/L,about 2.5 g/L to about 10.5 g/L, about 1.5 g/L to about 11.5 g/L, orabout 2 g/L to about 10 g/L of culture medium. In some embodiments, theculture medium comprises pyruvate at an amount of about 5 g/L to about15 g/L of culture medium, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15 g/L or any value or range therein. In some embodiments, theculture medium comprises pyruvate in an amount from about 5 g/L to about14.5 g/L, about 10 g/L to about 15 g/L, about 7.5 g/L to about 10.5 g/L,about 5.5 g/L to about 14.5 g/L, or about 8 g/L to about 10 g/L ofculture medium.

In some embodiments, the culture medium comprises a chemical bufferingsystem in an amount from about 1 g/L to about 4 g/L (e.g., about 1, 1.5,2, 2.5, 3, 3.5, or 4 g/L or any value or range therein) of culturemedium or about 10 mM to about 25 mM (e.g., about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM or any value or rangetherein). In some embodiments, the chemical buffering system includes,but is not limited to, sodium bicarbonate and/or HEPES. For example, insome embodiments, the culture medium comprises sodium bicarbonate in anamount from about 1 g/L to about 4 g/L of culture medium, e.g., about 1,1.5, 2, 2.5, 3, 3.5, or 4 g/L or any value or range therein. In someembodiments, the culture medium comprises sodium bicarbonate in anamount from about 1 g/L to about 3.75 g/L, about 1.25 g/L to about 4g/L, about 2.5 g/L to about 3 g/L, about 1.5 g/L to about 4 g/L, orabout 2 g/L to about 3.5 g/L of culture medium. In some embodiments, theculture medium comprises HEPES in an amount from about 10 mM to about 25mM, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 mM or any value or range therein. In some embodiments, theculture medium comprises HEPES in an amount from about 1 mM to about 25mM, about 10 mM to about 20 mM, about 12.5 mM to about 22.5 mM, about 15mM to about 20.75 mM, or about 10 mM to about 20 mM.

In some embodiments, the culture medium comprises one or more essentialamino acids in an amount from about 0.5 mM to about 5 mM (e.g., about0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mM or any value or rangetherein) or about 0.5, 1, 1.5, 2 mM to about 2.5, 3, 3.5, 4, 4.5, or 5mM. In some embodiments, one or more essential amino acids is arginineand/or cysteine. For example, in some embodiments, the culture mediumcomprises arginine in an amount from about 0.5 mM to about 5 mM, e.g.,about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mM or any value or rangetherein. In some embodiments, the culture medium comprises arginine inan amount from about 0.5 mM to about 4.75 mM, about 2 mM to about 3.5mM, about 0.5 mM to about 3.5 mM, about 1 mM to about 5 mM, or about 3.5mM to about 5 mM. For example, in some embodiments, the culture mediumcomprises cysteine in an amount from about 0.5 mM to about 5 mM, e.g.,about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mM or any value or rangetherein. In some embodiments, the culture medium comprises cysteine inan amount from about 0.5 mM to about 4,75 mM, about 2 mM to about 3.5mM, about 0.5 mM to about 3.5 mM, about 1 mM to about 5 mM, or about 3.5mM to about 5 mM.

In some embodiments, the culture medium comprises one or more vitaminsand/or cofactors in an amount from about 0.01 μM to about 50 μM (e.g.,about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6, 7, 8,9, 10, 12.5, 15,17.5, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075, or50 μM or any value or range therein) or about 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or0.9 μM to about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6 μM or about 0.02, 0.025, 0.05, 0.075, 1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM to about 12.5, 15, 17.5, 20, 25, 30,35, 40, 45, 46, 47, 48, 49, 49.025, 49.05, 49.075, or 50 μM. In someembodiments, one or more vitamins and/or cofactors includes, but is notlimited to, thiamine and/or riboflavin. For example, in someembodiments, the culture medium comprises thiamine in an amount fromabout 0.025 μM to about 50 μM, e.g., 0.025, 0.05, 0.075, 1, 1.5, 2, 3,4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 46, 47,48, 49, 49.025, 49.05, 49.075, or 50 μM or any value or range therein.In some embodiments, the culture medium comprises thiamine in an amountfrom about 0.025 μM to about 45.075 μM, about 1 μM to about 40 μM, about5 μM to about 35.075 μM, about 10 μM to about 50 μM, or about 0.05 μM toabout 45.5 μM. In some embodiments, the culture medium comprisesriboflavin in an amount from about 0.01 μM to about 3 μM, e.g., 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 μM or any value orrange therein. In some embodiments, the culture medium comprisesriboflavin in an amount from about 0.01 μM to about 2.05 μM, about 1 μMto about 2.95 μM, about 0.05 μM to about 3 μM, about 0.08 μM to about1.55 μM, or about 0.05 μM to about 2.9 μM.

In some embodiments, the culture medium comprises one or more inorganicsalts in an amount from about 100 mg/L to about 150 mg/L of culturemedium (e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or150 mg/L or any value or range therein) or about 100 mg/L to about 150mg/L of culture medium (e.g., about 100, 105, 110, 115, 120, 125, 130,135, 140, 145, or 150 mg/L or any value or range therein). In someembodiments, exemplary one or more inorganic salts is calcium and/ormagnesium. For example, in some embodiments, the culture mediumcomprises calcium in an amount from about 100 mg/L to about 150 mg/L ofculture medium, e.g., about 100, 105, 110, 115, 120, 125, 130, 135, 140,145, or 150 mg/L or any value or range therein. In some embodiments, theculture medium comprises arginine in an amount from about 100 mg/L toabout 125 mg/L, about 105 mg/L to about 150 mg/L, about 120 mg/L toabout 130 mg/L, or about 100 mg/L to about 145 mg/L of culture medium.In some embodiments, the culture medium comprises magnesium in an amountfrom about 0.01 mM to about 1 mM, e.g., about 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mM orany value or range therein. In some embodiments, the culture mediumcomprises magnesium in an amount from about 0.05 mM to about 1 mM, about0.01 mM to about 0.78 mM, about 0.5 mM to about 1 mM, about 0.03 mM toabout 0.75 mM, or about 0.25 mM to about 0.95 mM.

In some embodiments, the carbon source, chemical buffering system, oneor more essential amino acids, one or more vitamins and/or cofactors,and/or one or more inorganic salts is food grade.

In some embodiments, the culture medium is lactogenic culture medium,e.g., the culture medium further comprises prolactin (e.g., mammalianprolactin, e.g., human prolactin). For example, in some embodiments, theculture medium comprises prolactin (or prolactin is added) in an amountfrom about 20 ng/mL to about 200 ng/L of culture medium, e.g., about 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, or 200 ng/mL or any value or range therein. In some embodiments,the culture medium comprises prolactin (or prolactin is added) in anamount from about 20 ng/mL to about 195 ng/mL, about 50 ng/mL to about150 ng/mL, about 25 ng/mL to about 175 ng/mL, about 45 ng/mL to about200 ng/mL, or about 75 ng/mL to about 190 ng/mL of culture medium. Insome embodiments, the methods further comprise adding prolactin to theculture medium, thereby providing a lactogenic culture medium. In someembodiments, the prolactin is produced by a microbial cell and/or ahuman cell expressing a recombinant prolactin (e.g., a prolactincomprising a substitution of a serine residue at position 179 of theprolactin gene with aspartate (S179D), e.g., S179D-prolactin). In someembodiments, adding prolactin to the culture medium comprisesconditioning culture medium by culturing cells that express and secreteprolactin, and applying the conditioned culture medium comprisingprolactin to the basal surface of the monolayer of primary mammaryepithelial cells, the basal surface of the monolayer of the mixedpopulation, or the basal surface of the monolayer of live immortalizedmammary epithelial cells.

In some embodiments, the culture medium further comprises other factorsto improve efficiency, including, but not limited to, insulin, anepidermal growth factor, and/or a hydrocortisone. In some embodiments,the methods of the present invention further comprise adding otherfactors (e.g., insulin, an epidermal growth factor, and/or ahydrocortisone) to the culture medium, e.g., to improve efficiency.

Methods of Producing Cultured Milk Products

Disclosed herein, in certain embodiments, are methods of making acultured milk product. In some embodiments, the method comprisesculturing a live cell construct disclosed herein in a bioreactorcomprising a basal compartment and an apical compartment, wherein thebasal compartment comprises a culture media and the mammary cells secretthe cultured milk product into the apical compartment.

In some embodiments, the live cell construct comprises a scaffoldcomprising an upper surface and a lower surface and a polarizedmonolayer of live primary mammary epithelial cells, a continuouspolarized monolayer of a mixed population of live primary mammaryepithelial cells, mammary myoepithelial cells and mammary progenitorcells, and/or a continuous polarized monolayer of live immortalizedmammary epithelial cells having an apical surface and a basal surface,wherein the continuous polarized monolayer of live primary mammaryepithelial cells, the continuous polarized monolayer of the mixedpopulation of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells and/or the continuouspolarized monolayer of live immortalized mammary epithelial cells arelocated on the upper surface of scaffold

In some embodiments, the lower surface of the scaffold is adjacent tothe basal compartment. In some embodiments, the apical surface of thecontinuous polarized monolayer of live primary mammary epithelial cells,the continuous polarized monolayer of the mixed population of liveprimary mammary epithelial cells, mammary myoepithelial cells andmammary progenitor cells, and/or the continuous polarized monolayer oflive immortalized mammary epithelial cells is adjacent to the apicalcompartment. In some embodiments, the continuous polarized monolayer oflive primary epithelial mammary cells, the live primary epithelialmammary cells of the continuous polarized monolayer of the mixedpopulation of live primary mammary epithelial cells, mammarymyoepithelial cells and mammary progenitor cells, or the continuouspolarized monolayer of immortalized mammary epithelial cells secretesmilk through its apical surface into the apical compartment, therebyproducing milk in culture.

In some embodiments, the polarized monolayer of epithelial mammary cellsforms a barrier that divides the apical compartment and the basalcompartment, wherein the basal surface of the mammary cells are attachedto the scaffold and the apical surface is oriented toward the apicalcompartment.

In some embodiments, the basal compartment is adjacent to the lowersurface of the scaffold. In some embodiments, the basal compartmentcomprises a culture medium in fluidic contact with the basal surface ofthe polarized monolayer of mammary epithelial cells (e.g., the polarizedmonolayer of primary mammary epithelial cells, the polarized themonolayer of the mixed population, or the polarized monolayer of liveimmortalized mammary epithelial cells).

In some embodiments, the culture medium comprises a carbon source, achemical buffering system, one or more essential amino acids, one ormore vitamins and/or cofactors, and one or more inorganic salts.

In some embodiments, the bioreactor comprises an apical compartment thatis adjacent to the apical surface of the monolayer. In some embodiments,the apical compartment is adjacent to the upper surface of the scaffold.

In some embodiments, the total cell density of mammary cells in thebioreactor is at least 10¹¹ mammary cells. In some embodiments, thetotal cell density of mammary cells in the bioreactor is at least 10¹²mammary cells. In some embodiments, the total cell density of mammarycells in the bioreactor is at least 10¹³ mammary cells.

In some embodiments, the total cell density of mammary cells in thebioreactor is about 20 to 55 cells per 100 μm². In some embodiments, thetotal cell density of mammary cells in the bioreactor is about 20 cellsper 100 μm² In some embodiments the total cell density of mammary cellsin the bioreactor is 25 cells per 100 μm². In some embodiments, thetotal cell density of mammary cells in the bioreactor is about 30 cellsper 100 μm². In some embodiments, the total cell density of mammarycells in the bioreactor is about 35 cells per 100 μm². In someembodiments, the total cell density of mammary cells in the bioreactoris about 40 cells per 100 μm². In some embodiments, the total celldensity of mammary cells in the bioreactor is about 45 cells per 100μm². In some embodiments, the total cell density of mammary cells in thebioreactor is about 50 cells per 100 μm². In some embodiments, the totalcell density of mammary cells in the bioreactor is about 55 cells per100 μm².

In some embodiments, the total surface area of mammary cells within thebioreactor is at least about 1.5 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about 2m². In some embodiments, the total surface area of mammary cells withinthe bioreactor is at least about 2.5 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about 3m². In some embodiments, the total surface area of mammary cells withinthe bioreactor is at least about 4 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about 5m². In some embodiments, the total surface area of mammary cells withinthe bioreactor is at least about 10 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about 15m². In some embodiments, the total surface area of mammary cells withinthe bioreactor is at least about 20 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about 25m². In some embodiments, the total surface area of mammary cells withinthe bioreactor is at least about 50 m². In some embodiments, the totalsurface area of mammary cells within the bioreactor is at least about100 m². In some embodiments, the total surface area of mammary cellswithin the bioreactor is at least about 250 m². In some embodiments, thetotal surface area of mammary cells within the bioreactor is at leastabout 500 m².

In some embodiments, the bioreactor maintains a temperature of about 27°C. to about 39° C. (e.g., a temperature of about 27° C., 28° C., 29° C.,30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 35° C., 35.5° C., 36°C., 36.5° C., 37° C., 37.5° C., 38° C., 38.5° C. or about 39° C., or anyvalue or range therein, e.g., about 27° C. to about 38° C., about 36° C.to about 39° C., about 36.5° C. to about 39° C., about 36.5° C. to about37.5° C., or about 36.5° C. to about 38° C.). In some embodiments, thebioreactor maintains a temperature of about 37° C.

In some embodiments, the bioreactor has an atmospheric concentration ofCO₂ of about 4% to about 6%, e.g., an atmospheric concentration of CO₂of about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, or 6% or anyvalue or range therein, e.g., about 4% to about 5.5%, about 4.5% toabout 6%, about 4.5% to about 5.5%, or about 5% to about 6%). In someembodiments, the bioreactor has an atmospheric concentration of CO₂ ofabout 5%.

In some embodiments, the bioreactor has an atmospheric concentration ofCO₂ of about 4% to about 6%, e.g., an atmospheric concentration of CO₂of about 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, or 6% or anyvalue or range therein, e.g., about 4% to about 5.5%, about 4.5% toabout 6%, about 4.5% to about 5.5%, or about 5% to about 6%). In someembodiments, the bioreactor has an atmospheric concentration of CO₂ ofabout 5%.

In some embodiments, the method comprises monitoring the concentrationof dissolved O₂ and CO₂. In some embodiments, the concentration ofdissolved 02 is maintained between about 10% to about 25% or any valueor range therein (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25%). For example, in some embodiments, theconcentration of dissolved 02 is maintained between about 12% to about25%, about 15% to about 22%, about 10% to about 20%, about 15%, about20%, or about 22%. In some embodiments, the concentration of CO₂ ismaintained between about 4% to about 6%, e.g., a concentration of CO₂ ofabout 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, or 6% or any valueor range therein, e.g., about 4% to about 5.5%, about 4.5% to about 6%,about 4.5% to about 5.5%, or about 5% to about 6%). In some embodiments,the concentration of CO₂ is maintained at about 5%.

In some embodiments, the culture medium is exchanged about every day toabout every 10 days (e.g., every 1 day, every 2 days, every 3 days,every 4 days, every 5 days, every 6 days, every 7 days, every 8 days,every 9 days, every 10 days, or any value or range therein, e.g., aboutevery day to every 3 days, about every 3 days to every 10 days, aboutevery 2 days to every 5 days). In some embodiments, the culture mediumis exchanged about every day to about every few hours to about every 10days, e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to about every 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 days or any value or range therein. For example,in some embodiments, the culture medium is exchanged about every 12hours to about every 10 days, about every 10 hours to about every 5days, or about every 5 hours to about every 3 days.

In some embodiments, the method comprises monitoring the glucoseconcentration and/or rate of glucose consumption in the culture mediumand/or in the lactogenic culture medium. In some embodiments, theprolactin is added when the rate of glucose consumption in the culturemedium is steady state.

In some embodiments, the method further comprises applyingtransepithelial electrical resistance (TEER) to measure the maintenanceof the monolayer of epithelial cells. TEER measures a voltage differencebetween the fluids (e.g., media) in two compartments (e.g., between theapical and basal compartments), wherein if the barrier between thecompartments loses integrity, the fluids in the two compartments maymix. When there is fluid mixing, the voltage difference will be reducedor eliminated; a voltage difference indicates that the barrier isintact. In some embodiments, upon detection of a loss of voltage byTEER, a scaffold (e.g., a Transwell® filter, a microstructuredbioreactor, a decellularized tissue, a hollow fiber bioreactor, etc.) isreinoculated with additional cells and allowed time to reestablish abarrier (e.g., a monolayer) before resuming production of the culturedmilk product (e.g., milk production).

In some embodiments, the method further comprises collecting thecultured milk product from the apical compartment to produce collectedcultured milk product. In some embodiments, the collecting is via aport, via gravity, and/or via a vacuum. In some embodiments, a vacuum isattached to a port.

In some embodiments, the method further comprises freezing the collectedcultured milk product to produce frozen cultured milk product and/orlyophilizing the collected cultured milk product to produce lyophilizedcultured milk product.

In some embodiments, the method further comprises packaging thecollected cultured milk product, the frozen cultured milk product and/orthe lyophilized cultured milk product into a container.

In some embodiments, the method further comprises extracting one or morecomponents from the collected cultured milk product. Non-limitingexamples of components from the collected cultured milk product includemilk protein, lipid, carbohydrate, vitamin, and/or mineral contents. Insome embodiments, the components from the collected cultured milkproduct are lyophilized and/or concentrated to produce a lyophilized ora concentrated cultured milk product component product. In someembodiments, the components from the collected cultured milk product areconcentrated by, e.g., membrane filtration and/or reverse osmosis. Insome embodiments, the lyophilized or concentrated cultured milk productcomponent product is packaged in a container, optionally wherein thecontainer is sterile and/or a food grade container. In some embodiments,the container is vacuum-sealed. In some embodiments, the container is acanister, ajar, a bottle, a bag, a box, or a pouch.

Cultured Milk Products

Disclosed herein, in certain embodiments, are cultured milk products. Insome embodiments, the cultured milk product is a standardized, sterilecultured milk product. In some embodiments, the cultured milk product isfor nutritional use.

In some embodiments, the cultured milk product is produced by any methoddisclosed herein.

Breast milk contains low but measurable concentrations of environmentalcontaminants, health-harming chemicals from industry and manufacturingproducts that are widely spread in the environment. Environmentalcontaminants are partly secreted in breast milk. The contaminant levelsin breast milk reflect those in the mother's body and are thereforeideal for monitoring exposure levels. Toxic environmental contaminantscan be transferred from mother to infant via breastfeeding. Persistentorganic pollutants (POPs) are a family of lipophilic stable chemicalsthat bioaccumulate in adipose tissue and create a lasting toxic bodyburden. Breastfeeding provides a significant source of exposure to POPsearly in human life, the effects of which are unknown.

In some embodiments, the cultured milk product does not comprise or issubstantially free of one or more environmental contaminants. In someembodiments, the cultured milk product does not comprise or issubstantially free of persistent organic pollutants (POPs). In someembodiments, the cultured milk product does not comprise or issubstantially free of polychlorinated dibenzo-p-dioxins (PCDDs),polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs)and pesticides such as DDT.

Heavy metals such as mercury, lead, arsenic, cadmium, nickel, chromium,cobalt, zinc, and other potentially toxic metals that are dispersedthroughout the environment also have bioaccumulative features known toaccumulate in human milk and thus are of concern to the nursing infant.Metal in breast milk originates from exogenous sources, i.e., uptake viacontaminated air, food, and drinking water, and endogenous release alongwith essential trace elements. For example, lead and mercury are equallydispersed in the human food chain, and their impact on fetal developmentis heavily determined by the mother's diet and nutritional status. Theexposures to toxic metals have significant public health implication,even at small concentrations and acute exposures, these metals remaintoxic to humans. A nursing infant may be exposed to toxic metals in aperiod of highest susceptibility. Nursing infants may be exposed toheavy metals through breast milk in excess of what they should, andexposure may have health implication for the infants. For infants inparticular, these exposures may have adverse effect on the developingcentral nervous system, leaving a life-long defect on their cognitiveabilities.

In some embodiments, the cultured milk product does not comprise or issubstantially free of one or more heavy metals, such as arsenic, lead,cadmium, nickel, mercury, chromium, cobalt, and zinc. In someembodiments, the cultured milk product does not comprise or issubstantially free of arsenic. In some embodiments, the cultured milkproduct does not comprise or is substantially free of lead. In someembodiments, the cultured milk product does not comprise or issubstantially free of cadmium. In some embodiments, the cultured milkproduct does not comprise or is substantially free of nickel. In someembodiments, the cultured milk product does not comprise or issubstantially free of mercury. In some embodiments, the cultured milkproduct does not comprise or is substantially free of chromium. In someembodiments, the cultured milk product does not comprise or issubstantially free of cobalt. In some embodiments, the cultured milkproduct does not comprise or is substantially free of zinc. In someembodiments, the cultured milk product does not comprise or issubstantially free of arsenic, lead, cadmium, nickel, mercury, chromium,cobalt, and zinc.

Foreign allergenic proteins can be difficult to distinguish fromendogenous human milk proteins. Food proteins with allergenic potentialthat have been detected in human milk include hen's egg and peanutproteins. There are eight major food allergens, known as the big 8, thatare responsible for most of the serious food allergy reactions in theU.S. The big 8 list is comprised of milk, egg, fish, crustaceanshellfish, tree nuts, peanuts, wheat, and soybean allergens. Proteinsknown to cause egg allergy include ovomucoid, ovalbumin, and conalbumin.Peanuts proteins include arachin 6, arachin 3, conarachin, main allergenArah1, and arachin Arah2. As an example of maternal dietary proteintransportation to milk, it has been shown that the consumption of oneegg per day leads to higher concentrations of the chicken egg allergenovalbumin (OVA) in human milk compared to egg-avoiding mothers.

In some embodiments, the cultured milk product does not comprise or issubstantially free of one or more food allergens. In some embodiments,the cultured milk product does not comprise or is substantially free ofegg, fish, crustacean shellfish, tree nuts, peanuts, wheat, and soybeanallergens. In some embodiments, the cultured milk product does notcomprise or is substantially free of egg allergens. In some embodiments,the cultured milk product does not comprise or is substantially free offish allergens. In some embodiments, the cultured milk product does notcomprise or is substantially free of crustacean allergens. In someembodiments, the cultured milk product does not comprise or issubstantially free of tree nut allergens. In some embodiments, thecultured milk product does not comprise or is substantially free ofpeanut allergens. In some embodiments, the cultured milk product doesnot comprise or is substantially free of wheat allergens. In someembodiments, the cultured milk product does not comprise or issubstantially free of soybean allergens.

In some embodiments, the cultured milk product does not comprise or issubstantially free of arachin 6, arachin 3, conarachin, Arah1, andArah2.

In some embodiments, the cultured milk product does not comprise or issubstantially free of ovalbumin (OVA).

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

EXAMPLES Example 1

A cell culture system designed for the collection of milk should supportcompartmentalized secretion of the product such that the milk is notexposed to the media that provides nutrients to the cells. In the body,milk-producing epithelial cells line the interior surface of the mammarygland as a continuous monolayer. The monolayer is oriented such that thebasal surface is attached to an underlying basement membrane, while milkis secreted from the apical surface and stored in the luminalcompartment of the gland, or alveolus, until it is removed duringmilking or feeding. Tight junctions along the lateral surfaces of thecells ensure a barrier between the underlying tissues and the milklocated in the alveolar compartment. Therefore, in vivo, the tissue ofthe mammary gland is arranged such that milk secretion iscompartmentalized, with the mammary epithelial cells themselvesestablishing the interface and maintaining the directional absorption ofnutrients and secretion of milk.

The present disclosure describes a cell culture apparatus thatrecapitulates the compartmentalizing capability of the mammary glandthat is used to collect milk from mammary epithelial cells grown outsideof the body. Such an apparatus can include a scaffold to support theproliferation of mammary cells at the interface between twocompartments, such that the epithelial monolayer provides a physicalboundary between the nutrient medium and the secreted milk. In additionto providing a surface for growth, the scaffold provides spatial cuesthat guide the polarization of the cells and ensures the directionalityof absorption and secretion. This invention describes the preparation,cultivation, and stimulation of mammary epithelial cells in acompartmentalizing cell culture apparatus for the production andcollection of milk for nutritional use (see e.g., FIG. 1).

Preparation of mammary epithelial cells. Mammary epithelial cells areobtained from surgical explants of dissected mammary tissue (e.g.,breast, udder, teat), biopsy sample, or raw breastmilk. Generally, aftersurgical dissection of the mammary tissue, any fatty or stromal tissueis manually removed under aseptic conditions, and the remaining tissueof the mammary gland is enzymatically digested with collagenase and/orhyaluronidase prepared in a chemically defined nutrient media, whichshould be composed of ingredients that are “generally recognized assafe” (GRAS). The sample is maintained at 37° C. with gentle agitation.After digestion, a suspension of single cells or organoids is collected,either by centrifugation or by pouring the sample through a sterilenylon cell strainer. The cell suspension is then transferred to a tissueculture plate coated with appropriate extracellular matrix components(e.g., collagen, laminin, fibronectin).

Alternatively, explant specimens can be processed into small pieces, forexample by mincing with a sterile scalpel. The tissue pieces are platedonto a suitable surface such as a gelatin sponge or a plastic tissueculture plate coated with appropriate extracellular matrix.

The plated cells are maintained at 37° C. in a humidified incubator withan atmosphere of 5% CO₂. During incubation, the media is exchanged aboutevery 1 to 3 days and the cells are sub-cultured until a sufficientviable cell number is achieved for subsequent processing, which includespreparation for storage in liquid nitrogen; development of immortalizedcell lines through the stable transfection of genes such as SV40, TERT,or other genes associated with senescence; isolation of mammaryepithelial, myoepithelial, and stem/progenitor cell types by, forexample, fluorescence-activated cell sorting; and/or introduction into acompartmentalizing tissue culture apparatus for the production andcollection of milk for human consumption.

Cultivation of mammary epithelial cells for the production of milk. Milkfor nutritional use is produced by mammary epithelial cells isolated asdescribed above and cultured in a format that supports compartmentalizedsecretion such that separation between the nutrient medium and theproduct is maintained. The system relies on the ability of mammaryepithelial cells to establish a continuous monolayer with appropriateapical-basal polarity when seeded onto an appropriate scaffoldpositioned at the interface between the apical compartment, into whichmilk is secreted, and the basal compartment, through which nutrientmedia is provided (see, e.g., FIG. 2). Transwell® filters placed intissue culture plates, as well as bioreactors based on hollow fiber ormicrostructured scaffolds, for example, are used to support thesecharacteristics.

Following the isolation and expansion of mammary epithelial cells, thecells are suspended in a chemically defined nutrient medium composed offood-grade components and inoculated into a culture apparatus that hasbeen pre-coated with a mixture of extracellular matrix proteins, such ascollagen, laminin, and/or fibronectin. The cell culture apparatus is anydesign that allows for the compartmentalized absorption of nutrients andsecretion of product from a polarized, confluent, epithelial monolayer.Examples include hollow fiber and microstructured scaffold bioreactors(see, e.g., FIGS. 3 and 4, respectively). Alternatives include othermethods of 3-dimensional tissue culture, such as the preparation ofdecellularized mammary gland as a scaffold, repopulated with stem cellsto produce a functional organ in vitro, or collection of milk from thelumen of mammary epithelial cell organoids or “mammospheres” growneither in a hydrogel matrix or in suspension.

The apparatus includes sealed housing that maintains a temperature ofabout 37° C. in a humidified atmosphere of about 5% CO₂. Glucose uptakeis monitored to evaluate the growth of the culture as the cellsproliferate within the bioreactor. Stabilization of glucose consumptionindicates that the cells have reached a confluent, contact-inhibitedstate. The integrity of the monolayer is ensured using transepithelialelectrical resistance. Sensors monitor concentrations of dissolved O₂and CO₂ in the media at multiple locations. A computerized pumpcirculates media through the bioreactor at a rate that balances thedelivery of nutrients with the removal of metabolic waste such asammonia and lactate. Media can be recycled through the system afterremoval of waste using Lactate Supplementation and Adaptation technology(Freund et al. 2018 Int J Mol Sci. 19(2)) or by passing through achamber of packed zeolite.

Stimulation of milk production. In vivo and in cultured mammaryepithelial cells, the production and secretion of milk is stimulated byprolactin. In culture, prolactin can be supplied exogenously in thenutrient media at concentrations approximating those observed in thebody during lactation, e.g., about 20 ng/mL to about 200 ng/mL. Purifiedprolactin can be obtained commercially; however, alternative methods ofproviding prolactin or stimulating lactation are employed, includingexpression and purification of the recombinant protein from microbial ormammalian cell cultures. Alternatively, conditioned media prepared byculturing cells that express and secrete prolactin can be applied tomammary epithelial cell cultures to stimulate lactation. Bioreactors canbe set up in series such that media passing through a culture of cellsexpressing prolactin or other key media supplements is conditioned priorto exposure to mammary cells grown in a compartmentalizing cultureapparatus as described.

Other approaches to upregulate milk production and/or spare the use ofexogenous prolactin include molecular manipulation of the signalingpathways that are regulated by binding of prolactin to its receptor onthe surface of mammary epithelial cells, such as the following: (a)expression of constructs targeting the posttranslational modification ofprolactin; (b) expression of alternative isotypes of the prolactinreceptor; (c) expression of a chimeric prolactin receptor in which theextracellular domain is exchanged with the binding site for a differentligand; (d) introduction of a gene encoding a constitutively orconditionally active prolactin receptor or modified versions of itsdownstream effectors such as STATS or Akt; (e) knockout or modificationof the PER2 circadian gene; and/or (f) molecular approaches aimed atincreasing the rate of nutrient uptake at the basal surface of themammary epithelial monolayer.

Collection of milk. Secreted milk is collected continuously or atintervals through, for example, a port installed in the apicalcompartment of the culture apparatus. A vacuum is applied to the port tofacilitate collection and also contributes to the stimulation of furtherproduction. The collected milk is packaged into sterile containers andsealed for distribution, frozen or lyophilized for storage, or processedfor the extraction of specific components.

The present invention provides mammary epithelial cell cultures for theproduction of milk for nutritional use. In addition to human breastmilk, this method may be used to produce milk from other mammalianspecies, for example, for human consumption or veterinary use. Becauseit has not been previously possible to produce milk outside the body,this technology may result in novel commercial opportunities, inaddition to providing an alternative mode of production for existingproducts. The social and economic effects of the commercial developmentof this technology are broad and far reaching. Production of humanbreast milk from cultured cells may provide a means to address infantmalnutrition in food-scarce communities, provide essential nutrients topremature infants who are unable to breastfeed, and offer mothers a newoption for feeding their babies that provides optimal nutrition with theconvenience of infant formula. Production of cow or goat milk providesan opportunity to reduce the environmental, social, and animal welfareeffects of animal agriculture. The process described here addresses animportant gap in the emerging field of cellular agriculture andintroduces an opportunity to dramatically update the human food supplywithout compromising our biological and cultural attachment to the mostfundamental of our nutrition sources.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. Although the invention hasbeen described in detail with reference to preferred embodiments,variations and modifications exist within the scope and spirit of theinvention as described and defined in the following claims.

What is claimed is:
 1. A method of producing an isolated cultured milkproduct from mammary cells, the method comprising: (a) culturing a livecell construct in a bioreactor under conditions which produce thecultured milk product, said live cell construct comprising: (i) athree-dimensional scaffold having an exterior surface, an interiorsurface defining an interior cavity, and a plurality of pores extendingfrom the interior surface to the exterior surface; (ii) a matrixmaterial disposed on the exterior surface of the three-dimensionalscaffold; (iii) a culture media disposed within the interior cavity andin fluidic contact with the internal surface; and (iv) a confluentmonolayer of polarized mammary cells disposed on the matrix material,wherein the mammary cells are selected from the group consisting of:live primary mammary epithelial cells, live mammary myoepithelial cells,live mammary progenitor cells, live immortalized mammary epithelialcells, live immortalized mammary myoepithelial cells, and liveimmortalized mammary progenitor cells, and wherein the polarized mammarycells comprise an apical surface and a basal surface; and (b) isolatingthe cultured milk product.
 2. The method of claim 1, wherein thebioreactor comprises an apical compartment that is substantiallyisolated from the internal cavity of the live cell construct.
 3. Themethod of claim 1, wherein the basal surface of the mammary cells is influidic contact with the culture media.
 4. The method of claim 2,wherein the apical compartment is in fluidic contact with the apicalsurface of the mammary cells.
 5. The method of claim 4, wherein thecultured milk product is secreted from the apical surface of the mammarycells into the apical compartment.
 6. The method of claim 1, wherein theculture media substantially does not contact the cultured milk product.7. The method of claim 1, wherein total cell density of mammary cellswithin the bioreactor is at least 10¹¹.
 8. The method of claim 1,wherein total surface area of mammary cells within the bioreactor is atleast 1.5 m².
 9. The method of claim 1, wherein the matrix materialcomprises one or more extracellular matrix proteins.
 10. The method ofclaim 1, wherein the culturing is carried out at a temperature of about27° C. to about 39° C.
 11. The method of claim 1, wherein the culturingis carried out at an atmospheric concentration of CO₂ of about 4% toabout 6%.